Multiple Requirements of the Focal Dermal Hypoplasia Gene Porcupine During Ocular Morphogenesis

The American Journal of Pathology, Vol. 185, No. 1, January 2015

ajp.amjpathol.org

MOLECULAR PATHOGENESIS OF GENETIC AND INHERITED DISEASES Multiple Requirements of the Focal Dermal Hypoplasia Gene Porcupine during Ocular Morphogenesis

Elizabeth J. Bankhead,* Mary P. Colasanto,* Kayla M. Dyorich,* Milan Jamrich,yz L. Charles Murtaugh,x and Sabine Fuhrmann*

From the Departments of Ophthalmology and Visual Sciences,* John A. Moran Eye Center, and the Department of Human Genetics,x University of Utah, Salt Lake City, Utah; and the Departments of Molecular and Cellular Biologyy and Molecular and Human Genetics,z Baylor College of Medicine, Houston, Texas

Accepted for publication September 2, 2014. Wnt glycoproteins control key processes during development and disease by activating various downstream pathways. Wnt secretion requires post-translational modiﬁcation mediated by the O-acyl- Address correspondence to Sabine Fuhrmann, Ph.D., transferase encoded by the Drosophila porcupine homolog gene (PORCN). In humans, PORCN mutations Department of Ophthalmology cause focal dermal hypoplasia (FDH, or Goltz syndrome), an X-linked dominant multisystem birth defect and Visual Sciences, John A. that is frequently accompanied by ocular abnormalities such as coloboma, microphthalmia, or even Moran Eye Center, Rm S3180, anophthalmia. Although genetic ablation of Porcn in mouse has provided insight into the etiology of University of Utah Health defects caused by ectomesodermal dysplasia in FDH, the requirement for Porcn and the actual Wnt ligands Sciences Center, 65 Mario during eye development have been unknown. In this study, Porcn hemizygosity occasionally caused Capecchi Dr., Salt Lake City, ocular defects reminiscent of FDH. Conditional inactivation of Porcn in periocular mesenchyme led to UT 84132. E-mail: sabine. defects in mid- and hindbrain and in craniofacial development, but was insufﬁcient to cause ocular [email protected]. abnormalities. However, a combination of conditional Porcn depletion in optic vesicle neuroectoderm, lens, and neural crestederived periocular mesenchyme induced severe eye abnormalities with high penetrance. In particular, we observed coloboma, transdifferentiation of the dorsal and ventral retinal pigment epithelium, defective optic cup periphery, and closure defects of the eyelid, as well as defective corneal morphogenesis. Thus, Porcn is required in both extraocular and neuroectodermal tissues to regulate distinct Wnt-dependent processes during morphogenesis of the posterior and anterior segments of the eye. (Am J Pathol 2015, 185: 197e213; http://dx.doi.org/10.1016/j.ajpath.2014.09.002)

Focal dermal hypoplasia (FDH) or Goltz syndrome (OMIM secretion.9 Several human developmental disorders have been #305600) is an X-linked dominant syndrome resulting from linked to mutations in Wnt pathway components.10 defective development and interaction of ectodermal and Most FDH patients are heterozygous female with mosaic mesodermal tissues.1,2 FDH patients exhibit variable manifes- PORCN function, and the variable phenotypes are possibly tations of skin hypoplasia, hypodontia, skeletal abnormalities due to individual X-chromosome inactivation. Approximately (including limb and digit defects, as well as reduced bone den- 10% of FDH patients are male, with postzygotic mosaic sity), and defects in ocular, kidney, and abdominal wall devel- mutations. Studies in mouse revealed that PORCN is strictly opment. FDH is caused by mutations in the porcupine homolog required during gastrulation; thus, zygotic Porcn mutations are (Drosophila)gene(PORCN), which encodes for a highly conserved transmembrane O-acyltransferase localized to the Supported by NIH grants EY014954 (S.F.), R01-EY12505 (M.J.), and 3e5 endoplasmic reticulum. In mouse, PORCN-mediated pal- R21-OD010559 (L.C.M.), NIH Core Grant P30-EY014800 (Department of mitoylation is critical for trafﬁcking and signaling activity of Ophthalmology & Visual Sciences, University of Utah), and in part by an Wnt proteins, a family of highly conserved cysteine-rich gly- unrestricted grant from Research to Prevent Blindness, Inc. (New York, NY; Department of Ophthalmology & Visual Sciences, University of Utah). coproteins.6,7 Although Wnt-independent activity of PORCN 8 Disclosures: None declared. has been reported in some cases, a systematic analysis revealed Current address of M.P.C., Department of Human Genetics, University of that all Wnt proteins require palmitoylation by PORCN for their Utah, Salt Lake City, UT.

Copyright ª 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.09.002 Bankhead et al most likely lethal.11e14 Furthermore, zygotic deletion of the of small GTPases (RHO, RAC1, CDC42) and JNK, with paternal Porcn allele in mouse accurately recapitulates the participation of VANGL and DAAM (PCP pathway).39 In phenotypic mosaicism observed in female patients and results addition, noncanonical Wnt proteins such as WNT4, WNT5A, usually in perinatal lethality.11,15 Although FDH is considered and WNT11 can activate receptors other than Frizzled (eg, a rare disease, with a prevalence of 1:1,000,000 based on the ROR, RYK).40 Studies in frog and zebrafish indicate that number of observed live births, studies in mouse suggest that noncanonical Wnt signaling is essential for formation and/or e prenatal lethality may affect up to 98% of PORCN mutant maintenance of the eye field.38,41 44 In mouse, disruption of individuals, implying a prevalence of 1:25,000.15 Thus, FDH PCP effectors encoded by the Fuz, Wdpcp,andInt genes cause may affect embryonic survival much more significantly than anophthalmia (Fuz, Wdpcp) and coloboma (Int), respectively; has been acknowledged. however, the underlying cellular defects are unknown.45e47 PORCN is expressed in the developing mouse eye and Several Wnt proteins are robustly expressed in ocular and surrounding tissues, and FDH patients frequently exhibit periocular tissues, such as WNT2B, WNT3, WNT4, congenital eye defects, including microphthalmia, anoph- WNT5A, WNT5B, WNT7B, and WNT11 in the optic cup, thalmia, colobomata (iris, choroid, retina, and optic nerve), lens, or surface ectoderm and in the periocular mesen- aniridia, and pigment abnormalities.1,3,12,16 During normal eye chyme.48,49 Recent observations in chick, frog, and zebra- development, morphogenesis of the optic cup is a critical step fish demonstrate that WNT2B, WNT3A, WNT4, and that involves invagination of the distal optic vesicle and WNT11 regulate eye field formation and development of the overlying surface ectoderm. The resulting inner layer of the RPE and the lens.37,41,42,44,50,51 Interestingly, TGF-b optic cup develops into the neural retina, whereas the outer signaling can act cooperatively or adversely with Wnt pro- layer gives rise to the retinal pigment epithelium (RPE). The teins to regulate different processes of eye development, as ventral optic cup is connected to the forebrain by the optic recently shown in chick, suggesting that interference with stalk, and both the ventral optic cup and the stalk invaginate, Wnt signaling may also affect other pathways.37,50,51 To resulting in formation of the optic fissure. The peripheral rim date, however, no ocular defects resulting from deficiency of the optic cup differentiates into the ciliary body and iris in of particular Wnt proteins have been described in mammals. the postnatal mouse. The surrounding periocular mesenchyme Redundancy among Wnt ligands, crossregulation be- consists of multiple cell lineages, both neural crestederived tween noncanonical and canonical Wnt pathways, and and mesoderm-derived, and its interaction with the adjacent overlap of downstream components with other pathways neuroepithelium and lens ectoderm is critical for differentia- (and the lack of appropriate tools) have made it difficult to tion of the anterior segment, patterning of the RPE, and optic analyze the role of Wnt proteins, particularly those acting stalk. Developmental problems during these processes are via the noncanonical pathway in the developing mouse likely to cause the severe congenital ocular abnormalities eye.52 To gain insight into how ocular development is observed in FDH. affected in FDH and to understand the role of Porcn during Although the specific role of Porcn during eye develop- eye development in mammals, we disrupted Porcn in ocular ment is unknown, interference with downstream components and extraocular tissues in mouse. Our results demonstrate of Wnt pathways in mouse, zebrafish, chick, and frog has that PORCN is expressed in neuroectoderm, lens, and neural revealed that Wnt signaling is critical for diverse processes crestederived periocular mesenchyme and that it regulates during eye development. Wnt proteins bind to several surface closure of the optic fissure and eyelid, RPE differentiation, receptors, including the Frizzled family of transmembrane and corneal morphogenesis. proteins, and activate several different pathways. In mice and humans, 19 Wnt ligands and 10 Frizzled receptors have been Materials and Methods identified. The best characterized is the canonical Wnte b-catenin pathway, which functions through stabilization of Mice b–catenin, its translocation into the nucleus, and activation of TCF/LEF transcription factors. The role of the Wnteb- Animal handling and procedures were approved by the Uni- catenin pathway during eye development in vertebrates is versity of Utah Institutional Animal Care and Use Committee. often context- and species-dependent, with functions in Thegenerationofmicecarryingthefloxed Porcn allele coordinating retinal progenitor proliferation and differentia- [Porcnlox/lox; kindly provided by L. Charles Murtaugh (Uni- tion; development of the RPE, lens, ciliary body, and iris; and versity of Utah, Salt Lake City, UT)] has been described e ocular angiogenesis.17 38 For example, we and others have recently.13 For the purpose of distinction, male mice harboring shown that Wnteb-catenin signaling is required for differ- the floxed Porcn allele are referred to as Porcnlox/Y.Inall entiation of the RPE in the mouse optic cup, most likely by crosses, we maintained a mixed genetic background with direct interaction of TCF/LEF with enhancers of the key C57BL/6 and CD-1 mice (Charles River Laboratories Inter- regulatory genes Mitf and Otx2.20,21,25 national, Hollister, CA). Porcnlox/lox mice were crossed with In noncanonical Wnt signaling, activation of Frizzled re- Six3-Cre mice (kindly provided by Yasuhide Furuta, Riken ceptors leads to an increase in intracellular calcium and acti- Center for Developmental Biology, Kobe, Japan),53 Wnt1-Cre þ vation of PKC and CaMKII (Wnt/Ca2 pathway) or activation mice (Jackson Laboratory, Bar Harbor, ME),54 Rx3-Cre mice

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[kindly provided by Milan Jamrich (Baylor College of Med- Cell Signaling Technology), MITF (dilution 1:400; icine, Houston, TX)],55 ROSA26RLacZ mice (Jackson Labo- X1405M; Exalpha Biologicals, Shirley, MA), OTX2 ratory),56 and Axin2lacZ mice (Jackson Laboratory).57 Except (dilution 1:1500; AB9566; EMD Millipore), PAX2 (dilu- as otherwise indicated, wild-type (WT) littermates without a tion 1:100; Covance PRB-276P; BioLegend, Dedham, Cre allele were used as controls. MA), PAX6 (dilution 1:300; AB2237; EMD Millipore), Counting for timed pregnancies was started at embryonic PITX2 (dilution 1:1000; PA1020-100; Capra Science, day 0.5 (E0.5), the day a vaginal plug was detected. Embryos Angelholm, Sweden), b-tubulin class III (dilution 1:2000; were genotyped by PCR using limb or tail DNA with primer Covance PRB-435P; BioLegend), and VSX2 (dilution combinations as follows. The Porcn forward primer 50- 1:300; X1180P; Exalpha Biologicals). These were used in TGAGTGCTCAAATCCCAACC-30 and reverse primer 50- combination with the following secondary antibodies: goat CCAGCATGTGAAAATGTCAAC-30 generate Porcnwt (685 anti-rabbit, goat anti-mouse, or goat anti-rat, conjugated bp) and Porcnlox (762 bp) amplicons, and the reverse primer with Alexa Fluor 488, 568, or 647 (dilution 1:1000; Life 50-GTGTCCACCATGTGCATCTC-30 combines with the Technologies), donkey anti-goat conjugated with tetrame- same forward primer to produce the Porcn delta (PorcnD) thylrhodamine isothiocyanate (dilution 1:500; 705-025- amplicon (485 bp). Primers for Six3-(generic-)Cre were 147; Jackson ImmunoResearch, West Grove, PA), and forward 50-TCGATGCACGAGTGATGAG-30 and reverse donkey anti-sheep conjugated with tetramethylrhodamine 50-TTCGGCTATACGTAACAGGG-30;forSix3-(specific-) isothiocyanate (dilution 1:500; 713-165-003; Jackson Cre,forward50-CCCTTACGTCCTTCCTCCTC-30 and ImmunoResearch). For quantitative analysis, the number of reverse 50-ATGTTTAGCTGGCCCAAATG-30;forWnt1-Cre, caspase-3 and p-histone H3elabeled cells in the central and forward 50-TAAGAGGCCTATAAGAGGCGG-30 and reverse posterior optic cup was counted in alternating sagittal 50-ATCAGTCTCCACTGAAGC-30;forRx3-Cre, forward 50- sections for each eye at E11.5, and counts were analyzed GTTGGGAGAATGCTCCGTAA-30 and reverse 50-GTAT- using Student’s t-test. Whole-mount in situ hybridization CCCACAATTCCTTGCG-30;forSRY, forward 50-GCTGG- using digoxigenin-labeled Tbx5 and Vax2 riboprobes was GATGCAGGTGGAAAA-30 and reverse 50-CCCTCCG- performed as described previously.18 Except as otherwise ATGAGGCTGATATT-30;forROSAR26LacZ,forward50- indicated, at least three embryos were analyzed per geno- GGAGCGGGAGAAATGGATATG-30,reverse50-GCGAA- type, time point, and marker. GAGTTTGTCCTCAACC-30, and reverse 50-AAAGTCGC- Epifluorescence images were captured using an Olympus TCTGAGTTGTTAT-30. (Tokyo, Japan) XM10 camera on an upright Olympus BX51 microscope and were processed using Adobe Photoshop Histology, Immunohistochemistry, Quantitative CS3 software. Confocal images were captured using an Analysis, and in Situ Hybridization Olympus FV1000 system and were processed using ImageJ version 1.43u (NIH, Bethesda, MD) and Adobe Photoshop For histology, embryos were fixed in 4% formaldehyde, CS3 software. All other images were captured using an embedded in paraffin, sectioned at 5 mm and stained with Olympus MicroFire digital microscope camera U-CMAD3 hematoxylin and eosin according to standard procedures. mounted on the aforementioned BX51 microscope or on an For immunohistochemical analysis, embryo heads were Olympus SZX12 stereomicroscope. fixed in 4% paraformaldehyde, cryoembedded, and sec- m tioned (usually at 12 m). If necessary, cryostat sections Results were treated for antigen retrieval with hot citrate buffer (pH 6) or 1% Triton X-100. The following primary antibodies Porcn Hemizygosity Can Result in Diverse Ocular or markers were used for immunohistochemistry: BRN-3 Defects during Embryonic Development (dilution 1:50; sc-6026; Santa Cruz Biotechnology, Dallas, TX), caspase-3 (dilution 1:200; 559565; BD Pharmingen, In mouse, male Porcn-mutant embryos do not survive San Jose, CA), CDC42 (dilution 1:150; 2462; Cell beyond gastrulation, because of a failure in mesoderm for- Signaling Technology, Danvers, MA), cytokeratin 12 mation, and female heterozygous mutants typically die peri- (dilution 1:50; sc-17101; Santa Cruz Biotechnology), natally.11e13 To investigate the role of Porcn during ocular F-actin/phalloidin (dilution 1:500; A12379; Life Technol- development, we performed tissue-specificandtemporally ogies, Carlsbad, CA), b-galactosidase (b-gal) (dilution controlled inactivation, using females homozygous for a 1:5000; Cappel 855976; MP Biomedicals, Aurora, OH), b- floxed Porcn allele and males heterozygous for Six3-Cre.13,53 gal [dilution 1:750; a generous gift from Nadean Brown Six3-Cre is activated in the retina and ventral optic stalk at (University of California, Davis)], HES1 (dilution 1:1000; E9.0.53 Genotyping of extraocular tissues revealed that this a generous gift from Nadean Brown), phospho-histone H3 cross generated male conditional mutant embryos with two (dilution 1:1000; Upstate 06-570; EMD Millipore, Bill- distinct genotypes: Porcn mutants harboring a floxed Porcn erica, MA), phospho-c-JUN (dilution 1:500; 3270; Cell allele that developed normally (Porcnlox/Y;Six3-Cre; n Z 19) Signaling Technology), laminin (dilution 1:2000; ab30320; (Figure 1BandTable 1), and, unexpectedly, Porcn-mutant Abcam, Cambridge, MA), LEF1 (dilution 1:100; C12A5; embryos with a combination of floxed and delta alleles,

The American Journal of Pathology - ajp.amjpathol.org 199 Bankhead et al which in rare cases exhibited some developmental abnor- abnormalities can occur in the absence of Six3-Cre malities (PorcnD/lox/Y;Six3-Cre in Table 1). Because Six3-Cre (Figures 1Cand2A, and PorcnD/lox in Table 1), most likely can be expressed ectopically,58 this suggests postzygotic because of prezygotic deletion of the paternal Porcn allele by mosaic recombination of the floxed Porcn allele. ectopic CRE activity.15,58 In accord with previous Most PorcnD/lox/Y;Six3-Cre embryos seemed unaffected studies,11,13,15 we observed extraocular abnormalities with (Table 1) and when born showed normal life expectancy. variable frequency and severity, including abnormal cranio- D However, when Porcn /lox/Y;Six3-Cre were used as facial development and limbs with digit loss and/or fusion breeders, they recurrently generated female embryos exhib- (Figure 1C), as well as skin hypoplasia, open ventral body iting variable ocular and extraocular defects and harboring a wall, limb atrophy, tail defects (curly or short), and posterior combination of recombined and unrecombined Porcn alleles truncation (not shown). Female embryos harboring Six3-Cre (Porcnhet)(Figures 1 and 2 and Table 1). The observed in addition to recombined and unrecombined Porcn alleles exhibited similar defects; however, we could not distinguish between pre- or postzygotic deletion of Porcn (Figures 1E and 2C, and PorcnD/lox/þ;Six3-Cre in Table 1). Because the developmental defects are consistent across all genotypes, we show here representative examples of female offspring harboring a deleted Porcn allele with or without Six3-Cre, referred to hereafter as Porcnhet. Notably, we detected ocular abnormalities in Porcnhet embryos that have not been described previously and that may reflect, at least in part, extraocular Porcn deletion by ectopic CRE activity. Colobomata were detected in 10% of the embryos (n Z 60) (Figure 1, C and E and Table 1) and pigment defects of the optic cup with varying severity in up to 42% (Figure 1E and Table 1). To examine the pigment defects in more detail, we investigated whether differentiation of the RPE is disturbed. Normally, RPE specification and differentiation are regulated by the key regulatory transcription factors MITF and OTX2.59e61 Interference with MITF or OTX2 expression during early eye development results in microphthalmia and coloboma; the RPE transdifferentiates into retina because of a loss of RPE-specific morphology and gene expression, hyperproliferation, and ectopic up-

Figure 1 Retinal pigment epithelium (RPE) to retina transdifferentiation and closure defect of the optic fissure in heterozygous Porcn female embryos. A: Wild-type (WT) embryo at E15.5. B: Male embryo with Six3-Creemediated deletion of Porcn (Porcnlox/Y;Six3-Cre). C: Female embryo with heterozygous deletion of Porcn in the absence of Six3-Cre [genotype: PorcnD/lox (Porcnhet)] exhibiting syndactyly and absence of digits in the forelimb (asterisk), abnormal craniofacial development (arrowhead), and coloboma (arrow). D: WT eye at E13.5. E: Porcnhet embryonic eye at E13.5 with coloboma (arrowhead) and severe pigment loss in the dorsal RPE (arrow) (genotype: PorcnD/lox;Six3-Cre). F: Location of the sagittal sections in the remaining panels. GeN: Immmunohistological analysis of embryos at E13.5. G: OTX2 is expressed in the RPE (arrow) and, at this age, becomes normally expressed in progenitor cells and photoreceptor precursors in WT retina. H: In Porcnhet embryos, the dorsal RPE has lost widespread expression of OTX2 (arrow), but starts to acquire the retina- specific, dispersed pattern of OTX2 expression (genotype: PorcnD/lox;Six3- Cre). I: Expression of the retina-specific marker TUBB3 in differentiating neurons in WT retina. J: TUBB3 is misexpressed in the dorsal RPE of Porcnhet eyes (arrow) (genotype: PorcnD/lox). K: Expression of VSX2 in retinal progenitor cells in a control eye. L: In Porcnhet eyes, the dorsal RPE is multilayered and VSX2 is ectopically up-regulated (arrow) (genotype: PorcnD/lox;Six3-Cre). M: In control eyes, PAX6 expression is present in the retina, in the optic cup margins, RPE, anterior epithelium of the lens, and surface ectoderm. N: In Porcnhet eyes, PAX6 is expressed in all ocular tissues, including the dorsal RPE (arrow) (genotype: PorcnD/lox;Six3-Cre). Scale bars: 1 mm (AeC); 100 mm(D, E, GeN). Con, wild-type control.

200 ajp.amjpathol.org - The American Journal of Pathology Porcupine Gene in Ocular Morphogenesis

Table 1 Ocular and Extraocular Abnormalities in Porcn-Mutant Mice at Embryonic Age E12.5 to E18.0 RPE defects,* % Total Open Cranio-facial MHB embryos, Genotype OFCD, % Severe Moderate Mild eyelid,y % defects, % defects, % Normal, % No. Porcnlox/Y; Six3-Cre 0 0 0 0 0 0 0 100 19 PorcnD/lox/Y; Six3-Cre 0 0 0 7 13 3 3 90 29 Porcnhetz 10 5 18 42 43 28 3 42 60 PorcnD/lox 8 4 31 22 20 35 4 31 26 PorcnD/lox/þ; Six3-Cre 12 6 9 53 56 24 3 50 34 Porcnlox/Y; Wnt1-Cre 2 0 0 0 0 100 100 0 61 Porcnlox/Y; Rx3-Cre 13 0 20 50 0 95 17 0 30 Porcnlox/Y; Wnt1-Cre; Rx3-Cre 72 52 69 18 100 100 100 0 29 Porcnlox/þ; Wnt1-Cre; Rx3-Cre 9 0 9 82 0 0 0 22 23 WT 0 0 0 0 0 0 0 100 109

*Severe RPE defects: embryos with transdifferentiated RPE or almost complete loss of pigment (eg, Figure 1E). Moderate RPE defects: eyes with severe pigment gaps or reduction of pigment circumferentially (eg, Figure 2D). Mild RPE defects: eyes with small gaps or a subtle reduction of pigment circumferentially (eg, Figure 2A), analyzed between E15.5 and E18.0. yThe eyelid closure defect was determined at E16.5 to E18.0. zPorcnD/lox and PorcnD/lox;Six3-Cre combined. MHB, Mid- and hindbrain; NE, normal; OFCD, optic fissure closure defect; WT, wild-type. regulation of retina-specific genes (reviewed by Fuhrmann that Wnteb-catenin signaling is still active (Figure 2N). These et al62). In the developing eye, OTX2 is normally expressed observations suggest that the moderately affected RPE in both the RPE and retina; however, the expression pattern periphery does not undergo a complete transdifferentiation differs, reflecting different requirements in each tissue.61,63 In into retina, but rather acquires an intermediate fate. The overall Porcnhet embryos exhibiting severe pigment defects in the morphology of Porcnhet eyes exhibits other abnormalities; the dorsal optic cup, RPE-specific expression of OTX2 is down- vitreous is reduced (Figure 2, H and L), and the developing regulated (Figure 1, G and H). The affected RPE acquires a cornea appears thinner (Figure 2,H,L,andN).Furthermore, retinal fate by up-regulation of the retina-specific genes the ventral optic cup in Porcnhet embryos can exhibit hypo- Tubb3 and Vsx2 (Figure 1,IeL), whereas Pax6 is robustly plasia in the periphery (Figure 2,H,J,L,andN),evidentas expressed (Figure 1, M and N). These results indicate that loss of LEF1 expression (Figure 2N). large regions of the RPE in Porcnhet eyes can trans- differentiate into neural retina. Porcn Deletion in Multiple Tissues Is Required to Cause Porcnhet embryos also exhibited a range of less severe Ocular Defects pigment abnormalities in the optic cup periphery between E13.5 and E15.5, particularly as unpigmented patches or So far, our results showed that hemizygosity of Porcn can circumferential pigmentation loss (Figure 2 and Table 1). result in variable developmental ocular abnormalities with low Unpigmented patches can arise from missing tissue, as shown penetrance. We hypothesized that a further decrease in by interrupted laminin expression, separating two distinct PORCN expression would increase the incidence and con- areas of retinal tissue adjacent to the gap (Figure 2,AeC). sistency of ocular defects. In addition, several Wnt proteins are These gaps may be caused by asymmetrical growth and expressed in ocular tissues (optic cup, lens) and surrounding defective fusion of the optic cup margins. Circumferential periocular mesenchyme, but it is unclear in which tissue Wnt pigment loss ranges in severity, resulting from pigmentation expression is required.48,49 Because the periocular mesen- defects of the dorsal peripheral RPE (Figure 2D) and absence chyme provides important factors for optic fissure closure and of tissue in the ventral optic cup periphery (see Anterior RPE differentiation,65e67 we inactivated Porcn in neural Segment Abnormalities). Loss of dorsal pigmentation is crestederived periocular mesenchyme using Wnt1-Cre mice accompanied by a decrease in expression of the key regulatory (Porcnlox/Y;Wnt1-Cre54)(Supplemental Figure S1A). Porcn- genes Mitf and Otx2 (Figure 2,EeH). Interestingly, the retina- lox/Y;Wnt1-Cre embryos showed no ocular abnormalities (n Z specificgeneTubb3 is not ectopically up-regulated (Figure 2,I 61); however, we observed severe, fully penetrant defects in and J), in contrast to Vsx2 (Figure 2, K and L). The HMG box the mid- and hindbrain region (Figure 3,AandB,and transcription factor Lef1 can be a target and readout for active Table 1). This is consistent with deletion of other Wnt pathway canonical Wnt signaling in many tissues, including the components, including WLS (alias GPR177), Wnt1 and Wnt3, developing eye. Normally, LEF1 is expressed in very few cells Wnt5a, and b-catenin.68e72 In particular, the integral mem- in the embryonic retina but is robustly present in the peripheral brane protein GPR177 could be considered the closest RPE, corneal mesenchyme, and optic cup periphery functional Wnt pathway component to PORCN; it is required (Figure 2M).64 In Porcnhet embryos, LEF1 is strongly for proper trafficking and secretion of Wnt proteins and expressed in the dorsal, abnormal RPE periphery suggesting requires PORCN-mediated palmitoylation of Wnt proteins

The American Journal of Pathology - ajp.amjpathol.org 201 Bankhead et al for recognition.9,73,74 Mutant embryos with Wnt1-Cree mesenchyme and regulate proliferation, cell death, differentia- mediated disruption of Wls (alias Gpr177) fail to form the tion, and cell migration75 (reviewed by He and Chen76). isthmic organizer, resulting in abnormal mid- and hindbrain Porcnlox/Y;Six3-Cre embryos did not exhibit any obvious development.68 We therefore hypothesize that the isthmic ocular abnormalities (Figure 1B), and it is possible that the organizer may not be established properly in Porcnlox/Y;Wnt1- timing of Porcn inactivation in ocular tissues may be crit- Cre embryos; further studies are needed for confirmation. In ical. To examine the role of Porcn in ocular tissues earlier, addition, Porcnlox/Y;Wnt1-Cre embryos show abnormal we used another eye-specific Cre line, Rx3-Cre, that be- development of facial primordia, cleft palate, and a mild, fully comes active in the presumptive retina and RPE of the optic penetrant median cleft lip (Figures 3B, 4B, and 4G).13 Previous vesicle at E8.75 and can be ectopically expressed in the lens studies have demonstrated that inappropriate levels of Wnteb- vesicle (Supplemental Figure S1B).55 Rx3-Cre is also catenin and noncanonical Wnt signaling are associated with expressed in the epithelium of the medial nasal promi- cleft lip and palate in both humans and mice. Specifically, Wnt nence.55 In the present study, Porcnlox/Y;Rx3-Cre mutants proteins are expressed in the mesenchyme of the facial promi- (Figure 3C) occasionally exhibited ocular phenotypes such nences, in the oral ectoderm, and in the palatal shelf as mild circumferential loss of pigment and coloboma (Table 1). The penetrance was similar to that of Porcnhet embryos, suggesting that extraocular PORCN expression may be sufficient to prevent a higher incidence of ocular defects. In addition, most of the mutant embryos developed bilateral fusion defects in the upper lip and cleft palate, possibly because of Cre activity in the oral ectoderm (Figures 3C, 4C, and 4H and Table 1). So far, our observations suggested that PORCN may be required in multiple ocular and extraocular tissues. We therefore performed Porcn inactivation using a combination of Wnt1-Cre and Rx3-Cre lines (Supplemental Figure S1C). Indeed, Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos showed a much higher penetrance of ocular defects and, similar to Porcnlox/Y;Wnt1-Cre embryos, had full penetrance of abnormal mid- and hindbrain development, as well as cleft lip and palate (Figures 3D, 4D, 4I, and 5B and Table 1). In rare, extreme cases, the eyes were almost completely devoid of pigmentation (Figure 3H). Female embryos heterozygous

Figure 2 Porcnhet embryos exhibit pigment defects in the optic cup periphery at E13.5. A: Porcnhet eyes exhibit patches of depigmentation, often found temporally (arrowhead) (genotype: PorcnD/lox). B: Sagittal view of the ocular periphery shows continuous, basal laminin expression surrounding the lens and the retina and RPE in WT embryo. C: In Porcnhet embryos, unpigmented patches arise from tissue gaps evident by interrupted laminin expression (arrow) (same eye as in A). D: Colobomatous Porcnhet eye with circumferential decrease in pigment (arrow) (genotype: PorcnD/lox;Six3-Cre). E: Coronal view of expression of the RPE-specific protein MITF in WT dorsal RPE. F: In Porcnhet embryos, MITF expression is decreased in the RPE of the optic cup periphery (arrow) (same eye as in D). G: Coronal view of OTX2 expression in WT eye. H: OTX2 expression is slightly decreased in the dorsal RPE of Porcnhet embryos (arrow; same eye as in D). I: Expression of the retina-specific marker TUBB3 in WT retina. J: In Porcnhet eyes, TUBB3 is normally expressed in the retina and absent in the dorsal RPE periphery (arrow) (same eye as in D). K: Coronal expression of the retina-specific transcription factor VSX2 in WT retina. L: VSX2 is ectopically up-regulated in the dorsal RPE of Porcnhet embryos (arrow) (same eye as in D). M: In WT embryos at E13.5, LEF1 is expressed in the periphery of the retina and RPE, and in periocular mesenchyme. N: In Porcnhet embryos, LEF1 expression in the ventral optic cup (arrowhead) and in corneal mesenchyme (open arrowhead) is absent but robustly up-regulated in the dorsal RPE (arrow; same eye as in D). H, J, L, and N: Note the shortening of the ventral optic cup (arrowhead), reduction of the vitreous (asterisks), and thinning of the cornea (open arrowhead)inPorcnhet embryonic eyes. Scale bars: 200 mm(A and D); 100 mm(B, C, E, G, H, and I).

202 ajp.amjpathol.org - The American Journal of Pathology Porcupine Gene in Ocular Morphogenesis

Figure 3 Conditional Porcn inactivation in ocular and neural crestederived mesenchyme induces pigment abnormalities and transdifferentiation of the retinal pigment epithelium (RPE) at E15.5. AeD: Lateral views of WT (A), Porcnlox/Y;Wnt1-Cre (B), Porcnlox/Y;Rx3-Cre (C), and Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos (D). E: WT eye. FeH: Range of severities of ocular phenotypes found in Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos: colobomata (arrowhead) with largely circumferential pigment loss (F, arrow); more severe pigment loss dorsally (G); or, in rare cases, completely absent pigment (H). IeT: Coronal views. I and K: Expression of HES1 (red) in retinal progenitor cells and BRN-3 (green) in differentiating ganglion cells in WT retina, with WT ventral optic cup shown at higher magnification (K). J: In Porcnlox/Y;Wnt1-Cre;Rx3-Cre retina, expression of HES1 and BRN-3 shows a normal distribution. L: Higher magnification of the Porcnlox/Y;Wnt1-Cre;Rx3-Cre ventral optic cup reveals ectopic up-regulation of HES1 and BRN-3 in the RPE (arrow). The dotted line marks the apical borders of retina and transdifferentiated RPE; note optic stalk (asterisk) and reduced vitreous (arrowhead). M and O: Expression of OTX2 (green) in retinal progenitors and photoreceptor precursors and of VSX2 (red) in retinal progenitors in WT retina, with ventral optic cup shown at higher magnification (O). N: Expression of OTX2 and VSX2 appears normal in Porcnlox/Y;Wnt1-Cre;Rx3-Cre retina. P: Higher magnification of the Porcnlox/ Y;Wnt1-Cre;Rx3-Cre ventral optic cup reveals ectopic up-regulation of VSX2 and retinal OTX2 expression in the RPE (arrow); the dotted line marks the apical borders of retina and transdifferentiated RPE; note optic stalk (asterisk) and reduced vitreous (arrowhead). Q: LEF1 expression in WT retina. R: In Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos, LEF1 is down-regulated in the dorsal and ventral optic cup periphery (arrowheads) and in the ventral eyelid mesenchyme (asterisk). S: p-JUN expression in WT retina and lens. T: p-JUN in Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos appears normal. Scale bars: 1 mm (A and H); 200 mm(E); 100 mm(I, K, M, O, Q,andS).

The American Journal of Pathology - ajp.amjpathol.org 203 Bankhead et al

Figure 4 Craniofacial defects in conditional Porcn-mutant embryos. Gross (AeE) and histological (FeJ) images of control and mutant embryos at E15.5. Hematoxylin and eosin stain. A: Frontal view of WT embryo. B: Porcnlox/Y;Wnt1-Cre embryos have a mild cleft in the medial region of the upper lip (arrow). C: Porcnlox/Y;Rx3-Cre embryos have cleft lips (arrow). D: Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos exhibit craniofacial abnormalities including severe cleft lips (arrow). E: Normal craniofacial development in Porcnlox/þ;Wnt1-Cre;Rx3-Cre embryos. F: In control embryos, a uniform palate is formed (arrow). G: In Porcnlox/ Y;Wnt1-Cre embryos, the palatal shelves rise horizontally but do not fuse (arrow). H: Porcnlox/Y;Rx3-Cre embryos have cleft palate (arrow). I: In Porcnlox/Y;Wnt1- Cre;Rx3-Cre embryos, the palatal shelves do not rise above the tongue and they fail to fuse (arrow). J: Palatogenesis is normal in Porcnlox/þ;Wnt1-Cre;Rx3-Cre embryos (arrow). Scale bar Z 500 mm. for conditional Porcn inactivation using both Cre lines OTX2, and BRN-3, indicating that it undergoes trans- (Porcnlox/þ;Wnt1-Cre;Rx3-Cre) consistently and more differentiation into retina (Figure 3, K, L, O, and P). frequently (82%) exhibited mild RPE defects (Table 1). Furthermore, similar to Porcnhet eyes, the vitreous is Overall, our results demonstrate that the incidence and strongly reduced in Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes, and severity of pigment defects are markedly increased when the lens and retina are tightly attached to each other (Figure 3,L level of PORCN expression in retina, RPE, lens, and neural and P). Consistent with an effect of Porcn disruption on crestederived mesenchyme is further reduced. (Figure 3, Wnteb-catenin signaling, we observed a down-regulation FeH, and Table 1). In addition, affected eyes can appear of LEF1 in the optic cup periphery and ventral eyelid slightly microphthalmic (Figure 3E). Analysis of the eye mesenchyme (Figure 3, Q and R). p-JUN, the putative perimeter revealed that mutant eyes are 12% smaller than downstream effector of several signaling pathways control eyes: 87.84 5.74% in mutants (n Z 18 eyes from 9 (including noncanonical Wnt), appears to be unaffected in embryos) versus 100 7.98% in controls (n Z 30 eyes from ocular and extraocular tissues in Porcnlox/Y;Wnt1-Cre;Rx3- 16 embryos) (P < 0.0001). Further analysis of Porcnlox/ Cre embryos (Figure 3, S and T). Y;Wnt1-Cre;Rx3-Cre embryos revealed transdifferentiation of the dorsal RPE into retina, acquisition of intermediate fate The Defect in Closure of the Optic Fissure Is Associated or local loss of tissue (n Z 6 embryos at E15.5) (not shown), with Reduced Wnteb-Catenin Activity in the which is similar to Porcnhet eyes and results in a failure to Underlying Periocular Mesenchyme form the ciliary body and iris epithelium (iris hypoplasia; see Anterior Segment Abnormalities). Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos showed a high Recently, it has been shown that Fz5, Fz8, and Sfrp1/2 incidence of colobomata (72%) (Figure 3, F and G, and compound mutants exhibit accelerated generation of early- Table 1). During normal optic cup morphogenesis, the born ganglion cells and misregulation of HES1 expres- laterally growing edges of the RPE and retina at the margin sion, which is required for progenitor expansion during of the optic fissure align against each other to fuse and form neurogenesis in the embryonic mouse retina.77,78 We a continuous optic cup.79,80 The optic fissure forms as a therefore performed immunohistochemistry for markers la- ventral groove by asymmetric invagination extending from beling different populations of retinal progenitors (HES1, the vitreal side to the proximal junction with the forebrain, VSX2, OTX2) and early differentiating retinal cell types which allows mesenchymal cells to migrate inward and (BRN-3 for ganglion cells and OTX2 for photoreceptor form the hyaloid vasculature. Closure of the optic fissure in precursors) in Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes. We mouse starts at approximately E10.5 and is complete by observed no obvious difference in the developing central E12.5. The etiology of coloboma is complex. Coloboma can retina at E15.5 (Figure 3, J and N, and Supplemental result from genetic, extracellular signaling, or environmental Figure S2), which suggests that retinal neurogenesis at perturbations within the optic cup neuroepithelium and this stage proceeds largely normally, consistent with typical periocular mesenchyme. Many genes critical for closure of retinal expression of OTX2 and TUBB3 in Porcnhet eyes the optic fissure have been identified in humans and in an- (Figure 2, H and J). However, the ventral RPE ectopically imal models, including Pax2, Pax6, Vax2, Pitx2, JNK1/2, up-regulated the retina-specific expression of HES1, VSX2, and multiple Wnt pathway components21,77,81,82 (reviewed

204 ajp.amjpathol.org - The American Journal of Pathology Porcupine Gene in Ocular Morphogenesis by Gregory-Evans et al83 and Chang et al84); however, the Previous studies (eg, Westenskow et al,21 Martinez-Morales cellular mechanisms of the closure defects remain largely et al,61 Lee et al,85 and Tang et al86) have shown that genetic unresolved. To gain more insight into the possible mecha- models of RPE transdifferentiation can develop colobomata. nisms causing coloboma in Porcnlox/Y;Wnt1-Cre;Rx3- In Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos at E15.5, the Cre embryos, we examined eyes at E12.5, right after ventral RPE transdifferentiates (Figure 3, L and P), suggesting completion of optic fissure closure and when any closure that improper RPE differentiation could cause the closure defect first becomes obvious. Besides ocular defects, ab- defect in the optic fissure. However, we observed that RPE normalities in hindbrain and craniofacial development are differentiation appears to be normal at E12.5; MITF is present, detectable at this stage (Figure 5, A and B). The most and VSX2 expression is confined to the ventral retina in common type of coloboma in Porcnlox/Y;Wnt1-Cre;Rx3-Cre Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos (Figure 5,GeJ). mutants was characterized by a narrow gap in the ventral Furthermore, PAX2 and Vax2 are normally expressed in the optic cup, suggesting that the defect occurs late during the ventral optic cup of Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes closure process [9/11 (82%) embryos] (Figure 5,BeD). The (Figure 5,IeL). Expression of the dorsal patterning marker closure defect in Porcnlox/Y;Wnt1-Cre;Rx3-Cre mutants is Tbx5 is slightly reduced (Figure 5, M and N), which we also confirmed by persistent expression of the basement mem- observed in Porcnlox/Y;Rx3-Cre mutants (not shown) that brane component laminin (Figure 5, E and F). exhibit a low penetrance of colobomata (Table 1). Apoptotic

Figure 5 Patterning of the ventral optic cup in Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes. AeJ: Lateral (A and B) and sagittal (CeJ) views at E12.5. A: Control embryo. B: Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryo shows coloboma as a narrow gap in the ventral optic cup. In addition, defects in craniofacial (arrow) and mid- and hindbrain development (arrowhead) are detectable. C: Control eye. D: Porcnlox/Y;Wnt1-Cre;Rx3-Cre eye with open optic ﬁssure (arrow). E: Laminin (green) expression in the basement membrane surrounds ocular tissues in control eyes. Nuclei are counterstained with DAPI (blue). F: Persistent laminin expression in the ventral optic cup of Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes (arrow). G: Colabeling of control ventral optic cup with VSX2 (red) and MITF (green). H: Porcnlox/Y; Wnt1-Cre;Rx3-Cre ventral optic cup shows normal expression of VSX2 in retina and MITF in RPE. I: PAX2 expression (red) in ventral optic cup of control embryos. J: Porcnlox/Y;Wnt1-Cre;Rx3-Cre ventral optic cup exhibits normal PAX2 expression. KeN: Optic cups at E10.5. K: Lateral view of Vax2 mRNA expression in ventral optic cup of control embryos. L: Vax2 mRNA is expressed in the Porcnlox/Y;Wnt1-Cre;Rx3-Cre ventral optic cup (arrow). M: Tbx5 mRNA is expressed in the dorsal optic cup of control eyes. N: In Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes, Tbx5 mRNA expression is slightly reduced in the dorsal optic cup (arrow). Scale bar Z 100 mm.

The American Journal of Pathology - ajp.amjpathol.org 205 Bankhead et al cell death and proliferation are important processes during reporter line57 by detection of b-gal. In Porcnlox/Y;Wnt1- optic cup morphogenesis84,87,88; however, we observed no Cre;Rx3-Cre embryos, b-gal expression was reduced in the significant changes in the number of caspase-3 and p-histone mesenchyme underlying the optic fissure (n Z 5) (Figure 6,C H3elabeled cells in Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes dur- and D). Interestingly, b-gal expression was decreased in the ing the closure process (Supplemental Figure S3). Thus, RPE RPE as well, consistent with transdifferentiation into retina at differentiation, dorsoventral patterning, apoptosis, and prolif- E15.5. Furthermore, Wnteb-catenin activity is required for eration in the optic cup in Porcnlox/Y;Wnt1-Cre;Rx3-Cre em- maintenance of PITX2, a key transcription factor that is bryos show minimal changes, if any, and are unlikely to cause robustly expressed in the periocular mesenchyme (neural crest a defect in closure of the optic fissure. It is possible, however, and mesoderm) and required for anterior segment develop- that subsequent defects (eg, transdifferentiation of the ventral ment.65,89,90 Mutations in Pitx2 cause AxenfeldeRieger syn- RPE) could contribute to formation of a wider gap in the optic drome and can result in coloboma. However, PITX2 appears fissure at E15.5 (Figure 3). to be normally expressed in the periocular mesenchyme un- Next, we investigated whether changes in Wnt pathway derneath the optic fissure of Porcnlox/Y;Wnt1-Cre;Rx3-Cre activity occur in Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes. The embryos (Figure 6, E and F). Furthermore, we observed no number of LEF1-positive cells was reduced in periocular obvious changes in apical distribution of F-actin (Figure 6,G mesenchyme underlying the ventral optic cup of Porcnlox/ andH)orinexpressionofp-JUN(Figure 6, I and J). This Y;Wnt1-Cre;Rx3-Cre embryos (Figure 6, A and B). We also suggests that apicobasal polarity and some aspects of nonca- examined expression of Axin-2 in the Axin2lacZ knock-in nonical Wnt signaling appear to be normal in Porcnlox/Y;Wnt1- Cre;Rx3-Cre embryos. However, the effects on LEF1 expression and Axin2 reporter activation indicate that Wnteb- catenin signaling in the ventral optic cup and underlying periocular mesenchyme is compromised.

Abnormalities in Anterior Segment Development in Porcnlox/Y;Wnt1-Cre;Rx3-Cre Eyes

Further analysis of Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos showed that eyelid morphogenesis and some aspects of anterior segment development can be disturbed (Figure 7). Because of perinatal lethality, it is not possible to examine formation of the chamber angle and trabecular meshwork, which develop postnatally (reviewed by Cvekl and Tamm91). Normally, in the optic cup periphery at E17.5, the ciliary body starts to fold and some extension of the iris epithelium is detectable (Figure 7C). In Porcnlox/Y;Wnt1- Cre;Rx3-Cre eyes, pigmentation and early formation of ciliary body and iris are disturbed (Figure 7D), consistent

Figure 6 Porcn deficiency affects Wnteb-catenin activity in the periocular mesenchyme underlying the optic fissure (asterisks), shown in sagittal views at E12.5. A: In control embryos, LEF1 is expressed in the periocular mesenchyme underlying the ventral optic cup, particularly in two to three cell layers underneath the optic fissure (arrow). B: In Porcnlox/ Y;Wnt1-Cre;Rx3-Cre embryos, LEF1 is expressed in fewer cells underneath the optic fissure (arrow). C: b-Galactosidase expression in Axin2lacZ knock-in reporter embryos shows Axin-2 activation in the ventral retinal pigment epithelium (RPE) (arrowhead) and underlying mesenchyme (arrow). D: Activation of the Axin2 reporter is reduced (main image) or absent (inset) in the ventral RPE (arrowhead) and mesenchyme (arrow) in mutant embryos. E and F: PITX2 is expressed in the periocular mesenchyme underlying the optic fissure in WT embryos (E) and appears normal in Porcnlox/Y;Wnt1- Cre;Rx3-Cre embryos (F, arrow). G: Apical F-actin expression in the ventral optic cup of control embryos. H: F-actin expression shows a normal, continuous apical distribution in the Porcnlox/Y;Wnt1-Cre;Rx3-Cre ventral retina and RPE (arrow). I and J: Expression of phosphorylated c-JUN (p- JUN) in the ventral optic cup did not differ obviously between control (I) and Porcnlox/Y;Wnt1-Cre;Rx3-Cre (J) embryos. Scale bar Z 100 mm.

206 ajp.amjpathol.org - The American Journal of Pathology Porcupine Gene in Ocular Morphogenesis

Figure 7 Eyelid closure defect and abnormal corneal development in Porcnlox/Y;Wnt1-Cre;Rx3-Cre mutants. A: Control embryo at E17.5 with closed eyelid (arrow). B: Porcnlox/Y;Wnt1-Cre;Rx3-Cre mutant at E17.5 with open eyelid, coloboma, and pigment abnormalities in the optic cup periphery. C: Frontal section of the chamber angle of control eye at E17.5 with closed eyelid (arrowhead) and developing ciliary body and iris (asterisk). Hematoxylin and eosin stain. D: Porcnlox/Y;Wnt1-Cre;Rx3- Cre anterior segment at E17.5 shows growth-arrested eyelid folds (arrowhead), abnormal development of the peripheral retinal pigment epithelium (RPE) in the absence of ciliary body and iris (asterisk), and ectopic cells in the chamber angle (arrow). E: LEF1 expression in palpebral epidermis, mesenchyme (arrow), and conjunctival epithelium in upper eyelid of control eye at E15.5. F: In Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos, LEF1 expression in the palpebral epidermis and conjunctival epithelium of the upper eyelid is maintained, but is absent in the mesenchyme (arrow). G: Expression of LEF1 in the lower eyelid in control embryo. H: In the lower eyelid of Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos, LEF1 expression in the palpebral epidermis is decreased (right arrow), along with decreased expression in the mesenchyme (left arrow). I and K: Detection of p-JUN in the periderm, periderm extension (arrow), and conjunctival epithelium of upper (I) and lower (K) eyelids in control embryo at E15.5. J and L: In Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyelids at E15.5, p-JUN appears to be normal (arrows). M and N: Histological cross section of the cornea at E17.5 in control (M)andPorcnlox/Y;Wnt1-Cre;Rx3-Cre eyes (N). Corneal thickness is indicated by a bracket; the corneal endothelium is marked by an arrow. OeX: Embryos at E15.5. O: PAX6 (red) expression in the corneal epithelium of control eyes (arrow). Nuclei are counterstained with DAPI (blue). P: PAX6 is present in the corneal epithelium in Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes (arrow). Q and R: Expression of cytokeratin-12 in the corneal epithelium of control (Q)and Porcnlox/Y;Wnt1-Cre;Rx3-Cre (R) embryos shows no difference (arrows). S: In control eyes, LEF1 is expressed in the corneal mesenchyme (arrow) and epithelium (arrowhead). T: LEF1 is decreased in the Porcnlox/Y;Wnt1-Cre;Rx3-Cre corneal mesenchyme, except for some cells between lens and cornea (arrow). Reduced LEF1 expression in the corneal epithelium is variable (arrowhead). U: Robust PITX2 expression in control corneal mesenchyme (arrow). V: In Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes, PITX2 is present in the hypocellular corneal mesenchyme (arrow). W: The Rho GTPase CDC42 is robustly expressed in the corneal mesenchyme in control eyes (arrow). X: CDC42 expression is severely reduced in Porcnlox/Y;Wnt1-Cre;Rx3-Cre corneal mesenchyme (arrow). Scale bar Z 100 mm. with the patterning abnormalities of the optic cup margins observed in conditional mutants with a single Cre allele, observed at earlier ages (Figures 2 and 3). indicating that Porcn is required in multiple ocular surface The eyelid closure defect in Porcnlox/Y;Wnt1-Cre;Rx3-Cre tissues. Normally, between E11.5 and E15.5, the dorsal and embryos is completely penetrant (100%) (Table 1), and is not ventral periocular ectoderm invaginate, and the resulting

The American Journal of Pathology - ajp.amjpathol.org 207 Bankhead et al eyelid folds grow toward each other across the surface of the E11.5, and regulates neural crest migration and/or prolifer- eye. A projection of the outer, peridermal layer extends from ation during corneal morphogenesis and wound the eyelid margins across the cornea until the periderm ex- healing.100e103 We observed that CDC42 was strongly tensions meet and fuse. In Porcnlox/Y;Wnt1-Cre;Rx3-Cre expressed throughout the corneal mesenchyme in control mutants, the periocular ectoderm has invaginated at E15.5 eyes at E15.5, but was decreased in the Porcnlox/Y;Wnt1- (Figure 3); however, growth of the eyelid folds is arrested and Cre;Rx3-Cre cornea (Figure 7, W and X). Taken together, periderm extensions are not detectable (Figure 7,DeL). our observations suggest that Porcn deficiency results in LEF1 is normally present in conjunctival epithelial cells, corneal hypoplasia due to defects in proliferation, survival, palpebral epidermal cells, and in mesenchymal cells, and/or migration of the periocular mesenchyme. consistent with previous studies showing Axin2 or Tcf/Lef reporter expression in developing eyelids (Figure 7, E and Discussion G).92e95 In Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos, mesenchymal expression of LEF1 is reduced or absent in both in Our present findings show that Porcn depletion during optic upper and lower eyelids (Figure 7, F and H). In addition, cup morphogenesis leads to closure defects of the optic LEF1 expression is decreased in palpebral epidermal cells of fissure and eyelids, a hypocellular cornea, and a range of RPE the lower eyelid (Figure 7H). We also examined p-JUN defects. Unless Porcn is completely inactivated in most of the expression in the periderm and its extension, because it is ocular and extraocular tissues, these defects are highly vari- 96 required for eyelid closure. p-JUN is detectable in the able and less penetrant. This is reminiscent of ocular abnor- lox periderm of both upper and lower eyelids in Porcn / malities found in female FDH patients, who exhibit mosaic Y;Wnt1-Cre;Rx3-Cre eyes (Figure 7,IeL). Overall, these deletion of PORCN due to stochastic X-chromosome inac- results indicate that LEF1 expression in the eyelid mesen- tivation. Approximately 40% of FDH patients exhibit chyme is significantly affected by disruption of Porcn. ophthalmological findings, including microphthalmia, During normal morphogenesis of the cornea (approxi- anophthalmia, coloboma (of the eyelid, iris, choroid, retina, mately E12 in mouse), the periocular mesenchyme responds and optic nerve), aniridia, and hypopigmentation.1,2,16 Here, to cues from the lens and migrates into the space between we have shown that deletion of Porcn in multiple tissues lens vesicle and surface ectoderm, which matures into the mimics several aspects of FDH with high incidence, and our 91 corneal epithelium (reviewed by Cvekl and Tamm ). analysis provides insight into some of the mechanisms by Migrating mesenchymal cells either undergo mesenchymal- which Porcn regulates eye development. to-epithelial transition and differentiate into corneal endothelium or differentiate into keratocytes, which produce the Porcn Deficiency Causes RPE Abnormalities extracellular matrix of the corneal stroma. In Porcnlox/ Characteristic of Atypical Colobomata and Aniridia Y;Wnt1-Cre;Rx3-Cre eyes, the cornea separates from the lens (Figure 7, D and N), suggesting that maturation of the Porcn deficiency leads to loss of pigmentation in the optic corneal endothelium proceeds normally. However, similar cup periphery, and we identified tissue gaps or iris hypo- to Porcnhet eyes at E13.5 (Figure 2), we observed a thinning plasia as one possible cause. These gaps could be thought of of the cornea in Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes, caused as atypical iris colobomata, because they arise outside of the by a severe decrease in cell number (Figure 7, M and N). inferonasal quadrant harboring the optic fissure. Atypical The corneal epithelium is correctly specified in Porcnlox/ colobomata can occur independently from a closure defect Y;Wnt1-Cre;Rx3-Cre eyes, because PAX6 and cytokeratin- in the optic fissure and are also associated with anterior 12 are robustly expressed at E15.5 (Figure 7,OeR). This segment disorders such as aniridia and AxenfeldeRieger is consistent with previous studies indicating that Wnteb- syndrome (reviewed by Chang et al84). Circumferential catenin activity is not required during differentiation of the pigment defects are accompanied by tissue hypoplasia in corneal epithelium.97,98 In the corneal mesenchyme, Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes, reminiscent of aniridia. expression of LEF1 is markedly down-regulated, indicating These abnormalities are consistent with a role of Wnteb- reduced Wnteb-catenin activity (Figure 7, S and T). catenin signaling in differentiation of RPE, iris epithelium, However, similar to expression at E12.5, the Wnteb-cat- and ciliary body in the optic cup.24,27,30 Deletion of b-catenin target PITX2 is not affected by Porcn depletion; in enin in the optic cup periphery attenuates growth and Porcnlox/Y;Wnt1-Cre;Rx3-Cre eyes, PITX2 is expressed in patterning of the ciliary margin, accompanied by a short- the hypocellular corneal mesenchyme at E15.5 (Figure 7,U ened optic cup.24,30 Similarly, combined disruption of the and V). This suggests that differentiation of the hypocellular secreted Wnt modulators sFRP-1 and sFRP-2 (which are corneal mesenchyme at this age proceeds normally, despite required here for extracellular spreading and availability of reduced LEF1 expression. Furthermore, Rho GTPases can Wnt proteins) leads to identical defects.19,78 act as effectors of noncanonical Wnt signaling to regulate A considerable number of Porcnlox/Y;Wnt1-Cre;Rx3-Cre migration of the developing neural crest (reviewed by eyes exhibit severe transdifferentiation involving major Mayor and Theveneau99). Specifically, the GTPase CDC42 parts of the proximal RPE; to date, this has not been described is robustly expressed in the corneal mesenchyme, starting at in humans. Previous studies, including our own, have

208 ajp.amjpathol.org - The American Journal of Pathology Porcupine Gene in Ocular Morphogenesis demonstrated that a complex of b-catenineTCF/LEF directly expression, consistent with a role of Fz5 and Fz8 in transactivates enhancers of the RPE key regulatory genes neuronal polarity and possibly noncanonical Wnt signaling Mitf and Otx2; thus, deletion of b-catenin in the RPE leads during neural development.77,105 However, it has not been directly to a loss of MITF and OTX2, which are strictly determined whether apical junction formation is already required for RPE development.20,21,25,29 Our present findings impaired during closure of the optic fissure in triallelic Fz5/ provide further evidence that it is the signaling function of b- Fz8 mutants.77 Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos do catenin that is required to promote RPE differentiation. not exhibit obvious defects in retinal neurogenesis and Interestingly, our findings also suggest that Wnt proteins HES1 expression, and apicobasal polarity appears to be secreted from multiple tissues are required for Wnteb-cat- normal in the ventral optic cup. One possibility is that FZ5 enin activation in the RPE, because Porcn depletion in per- and FZ8 are activated by ligands independently of Wnt iocular mesenchyme or optic neuroepithelium or lens alone is proteins. On the other hand, there is increasing evidence that not sufficient to cause transdifferentiation of RPE into retina. interference with noncanonical Wnt signaling can result in We observed that, in rare cases, transdifferentiation ectopic activation of Wnteb-catenin signaling,52,106e109 involving large regions of the posterior RPE resulted in and disruption of the canonical Wnt antagonists Dkk-1 extreme RPE defects during optic cup formation (Figure 3H), and Axin-2 results in coloboma110 (A.A. and S.F., unpub- possibly because of an earlier onset of Porcn inactivation. It is lished data). It is possible, therefore, that colobomata in conceivable that this might later lead to severe microphthalmia; triallelic Fz5/Fz8 mutants result from ectopic activation of however, we could not explore this because of perinatal Wnteb-catenin signaling. lethality. Furthermore, Porcn depletion before the optic vesicle Disruption of the Wnt coreceptor LRP6 causes severe stage could result in extreme microphthalmia or anophthalmia. coloboma and dorsoventral patterning defects; specifically, Noncanonical Wnt signaling is required before optic vesicle the dorsal domain of the optic cup is dramatically reduced, formation in zebrafish and frog, either to suppress canonical as shown by loss of Tbx5 and expansion of Vax2 expres- Wnt pathways and/or to promote eye-specific gene expression.82 By contrast, in Porcnlox/Y;Wnt1-Cre;Rx3-Cre em- sion41,43,44 (reviewed by Fuhrmann104), and mutation of PCP bryos, colobomata are less severe, characterized usually by a effector genes such as Fuz, Int,orWdpcp results in micro- small gap ventrally, and dorsoventral patterning appears phthalmia or anophthalmia in mouse.45e47 Thus, the occur- largely normal. Although disruption of Porcn is expected to rence of FDH with severe microphthalmia or anophthalmia interfere with all Wnt pathways, this difference could be could be explained by an early role of noncanonical Wnt explained by unexpected, PORCN-independent secretion of signaling during eye development. some Wnt proteins. Another possibility is that Porcn deletion in our Porcnlox/Y;Wnt1-Cre;Rx3-Cre embryos is not Porcn Regulates Closure of the Optic Fissure complete. Interestingly, recent studies have identified novel, highly context-dependent functions of LRP6; although it is Similar to FDH in humans, in mouse the loss or reduction of required to activate Wnteb-catenin signaling, LRP6 mod- PORCN results in a failure of the optic fissure to close. We ulates the noncanonical Wnt/PCP pathway during mouse found that the neuroepithelium in the ventral optic cup and heart morphogenesis and neural tube closure.106,107 These underlying periocular mesenchyme differentiated properly and other studies show that the outcomes of LRP6 and Wnt up to E12.5, because several genes critical for closure of the signaling are highly context-dependent and can involve optic fissure (eg, Vax2, Pax2, Mitf, and Pitx2) were nor- antagonistic or coregulative action of both Wnteb-catenin mally expressed. Maintenance of PITX2 expression is and noncanonical Wnt pathways. Thus, it is possible that transiently dependent on Wnteb-catenin signaling,89 and so Lrp6 mutants may exhibit gain of function of noncanonical the normal expression pattern of PITX2 in Porcnlox/Y;Wnt1- Wnt signaling during closure of the optic fissure, and we Cre;Rx3-Cre mesenchyme was unexpected. This suggests hypothesize that this could exacerbate defects in optic cup that the temporal requirement for Wnteb-catenin activity morphogenesis. may have passed already. Furthermore, neural crestespe- cific inactivation of b-catenin causes abnormalities in the Defects in Eyelid Closure and Corneal Morphogenesis optic stalk and eye positioning; however, it is not clear on Porcn Disruption whether the optic fissure closes properly.89 Further studies are needed to determine whether the closure defects results FDH can manifest with coloboma of the eyelids, and our ob- from impaired Wnteb-catenin activity in the mesenchyme. servations in Porcn mutants are consistent with a role of Wnt Disruption of the Wnt receptor Frizzled-5 (encoded by signaling during eyelid morphogenesis and closure.13 LEF1 Fz5) during early embryonic development occasionally expression in the eyelid mesenchyme and eyelid epithelium is causes coloboma, and the incidence of coloboma increases decreased, indicating compromised Wnteb-catenin signaling. when one allele of the closely related Fz8 is additionally Defects can manifest as permanent open eyelids or delayed removed.77,81 These triallelic Fz5/Fz8 mutants exhibit closure, and disruption of several Wnteb-catenin and nonca- retinal neurogenesis defects that are attributed to abnormal nonical pathway genes (eg, Tcf3, Dkk2, Lrp6, Vangl2, Fz3, formation of retinal apical junctions and defective HES1 Fz6, Celsr1,andPtk7) causes eyelid closure abnormalities.95,98

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